Similarity of mouse perivascular and brown adipose tissues and their resistance to diet-induced inflammation - PubMed (original) (raw)

Similarity of mouse perivascular and brown adipose tissues and their resistance to diet-induced inflammation

Timothy P Fitzgibbons et al. Am J Physiol Heart Circ Physiol. 2011 Oct.

Abstract

Thoracic perivascular adipose tissue (PVAT) is a unique adipose depot that likely influences vascular function and susceptibility to pathogenesis in obesity and the metabolic syndrome. Surprisingly, PVAT has been reported to share characteristics of both brown and white adipose, but a detailed direct comparison to interscapular brown adipose tissue (BAT) has not been performed. Here we show by full genome DNA microarray analysis that global gene expression profiles of PVAT are virtually identical to BAT, with equally high expression of Ucp-1, Cidea, and other genes known to be uniquely or very highly expressed in BAT. PVAT and BAT also displayed nearly identical phenotypes upon immunohistochemical analysis, and electron microscopy confirmed that PVAT contained multilocular lipid droplets and abundant mitochondria. Compared with white adipose tissue (WAT), PVAT and BAT from C57BL6/J mice fed a high-fat diet for 13 wk had markedly lower expression of immune cell-enriched mRNAs, suggesting resistance to obesity-induced inflammation. Indeed, staining of BAT and PVAT for macrophage markers (F4/80 and CD68) in obese mice showed virtually no macrophage infiltration, and FACS analysis of BAT confirmed the presence of very few CD11b(+)/CD11c(+) macrophages in BAT (1.0%) compared with WAT (31%). In summary, murine PVAT from the thoracic aorta is virtually identical to interscapular BAT, is resistant to diet-induced macrophage infiltration, and thus may play an important role in protecting the vascular bed from inflammatory stress.

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Figures

Fig. 1.

Fig. 1.

Cidea and Ucp-1 are highly expressed in interscapular brown adipose tissue (BAT) and perivascular adipose from the aortic arch (PVAT) independently of obesity. A, B, C, and D: Cidea, Ucp-1, Acrp30, and Ppar_γ_2 expression in normal diet (ND) and high-fat diet (HFD) mice. Quantitative PCR was performed on total RNA isolated from inguinal (SAT), epididymal (VAT), BAT, and PVAT. Expression levels were calculated with the 2−ΔΔCt method using 36b4 as the reference gene and normalized to expression in SAT from ND conditions. Ucp-1 expression is shown in log10 scale. AU, arbitrary units. ND: n = 12 white; HFD: n = 12, black. Results are means ± SE. ***P < 0.001 vs. SAT ND; #P < 0.05. ND vs. HFD of the same fat depot using two-way ANOVA and the Bonferonni correction; P < 0.05 vs. SAT ND.

Fig. 2.

Fig. 2.

PVAT appears morphologically similar to BAT. Fat was harvested from SAT (A and B), VAT (C and D), interscapular BAT (E and F), and PVAT (G–J) from the lesser curvature of the aortic arch from ND and HFD fed mice and then fixed in formalin. Tissues were stained with hematoxylin and eosin and visualized at ×25. Images G and H are low magnification images (×6.3) of I and J. Ao, aortic lumen.

Fig. 3.

Fig. 3.

Transmission electron microscopy reveals many similarities between PVAT and BAT. Sections of brown (A and B) and perivascular adipose (C and D) were taken and stained with osmium tetroxide. In ND conditions, brown (A) and perivascular adipose (C) appear very similar with mulitilocular lipid droplets and abundant mitochondria. Two prominent changes were noted in high-fat feeding conditions; lipid droplets in BAT, but not PVAT, lose their avidity for osmium textroxide (B), and mitochondria become swollen with unfolded cristae (D); the latter effect was more prominent in perivascular adipose but also observed in brown adipose after a longer duration of HFD. LD, lipid droplet; M, mitochondria; EC, endothelial cell; RBC, red blood cell. Magnification: ×7900; scale bar = 2 μm.

Fig. 4.

Fig. 4.

Microarray analysis reveals that PVAT is more similar to BAT than SAT or VAT. Microarray Computational Environment (MACE) database was queried in ND and HFD conditions for genes with a >2.0 fold change in expression at P < 0.05 level of significance between adipose depots. In ND conditions, PVAT was very similar to BAT, with differential expression of only 228 genes (0.79% of genes); in contrast, expression of 1,229 genes was differentially regulated when comparing PVAT and SAT (4.2% of genes). After high-fat feeding, PVAT became more similar to white adipose, as the number of genes differentially regulated between PVAT and SAT was reduced to 855 (2.9% of genes). White arrows, upregulated; black arrows, downregulated.

Fig. 5.

Fig. 5.

PVAT and BAT are resistant to inflammation after 13 wk of HFD. SAT (A and B), VAT (C and D), BAT (E and F), and PVAT (G and H) from the aortic arch was harvested from lean and obese mice (n = 3 per group). Samples were fixed in 4% formalin, sectioned, and stained with a rat anti-mouse F4/80 primary antibody (ABd Serotec). Staining was visualized with a horseradish-peroxidase-linked rabbit anti-rat secondary antibody. Abundant macrophages were seen predominantly in VAT, but also SAT, forming crown-like structures (arrowheads). No macrophages were seen in BAT or PVAT (magnification: ×25).

Fig. 6.

Fig. 6.

Perivascular and brown adipose tissue are resistant to inflammation after 20 wk of HFD. A second cohort of mice was continued on HFD for 20 wk. SAT (A and B), VAT (C and D), BAT (E and F), and PVAT (G and H) from the aortic arch was harvested from lean and obese mice (n = 3 per group). Samples were fixed in 4% formalin, sectioned, and stained with a rat anti-mouse F4/80 primary antibody (ABd Serotec). Staining was visualized with a HRP linked rabbit anti-rat secondary antibody. Again, abundant macrophages were seen in VAT (D). No macrophages were seen in BAT (F) or PVAT (H) despite distortion and enlargement of lipid droplet morphology (magnification: ×25).

Fig. 7.

Fig. 7.

BAT is resistant to inflammation after 11 and 20 wk of HFD. A: after 11 wk of HFD, the stromal vascular fraction was isolated from VAT and BAT and stained with CD31-PE, CD11b-PerCp Cy5.5, and CD11c-PE Cy7. Samples were analyzed on a LSRII flow cytometer, gating for CD31-negative cells. High-fat feeding resulted in a significant increase in the percentage of CD11b- and CD11c-positive cells in the SVF of visceral but not brown adipose (31.7 vs. 1.03%). B: similar results were obtained after 20 wk of HFD. Results represent 3 independent experiments.

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